Mechanisms and modulation of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced Ca2+ mobilization in human neutrophils

The effect of fMLP (N-formyl-methionyl-leucyl-phenylalanine), a neutrophil-stimulating bacterial peptide, on Ca2+ mobilization in human neutrophils was examined using fura-2 as a Ca2+ indicator. fMLP (10 nM-10 muM) increased [Ca2+](i) concentration-dependently. The [Ca2+](i) signal comprised an initial rise followed by a gradual decay and a sustained phase. External Ca2+ removal partly decreased the signal. La3+ (50 muM) pretreatment mimicked the effect of Ca2+ removal. In Ca2+-free medium. pretreatment with 1 muM thapsigargin tan endoplasmic reticulum Ca2+ pump inhibitor) prevented 10 muM fMLP from increasing [Ca2+](i); whereas 1 muM thapsigargin still significantly increased [Ca2+](i) after pretreatment with 10 muM FMLP. Addition of 3 mM Ca2+ induced a concentration-dependent [Ca2+](i) increase after pretreatment with fMLP in Ca2+-free medium. This Ca2+ entry was partly inhibited by econazole (25 muM). SKF96365 (50 muM). and a phospholipase A, inhibitor (aristolochic acid; 20 muM) The fMLP (10 muM)-induced Ca2+ release was abolished by inhibiting phospholipase C with 2 muM U73122, The fMLP-induced [Ca2+](i) increase was inhibited by 25% by pretreatment with 10 nM phorbol ester to activate protein kinase C but was augmented by 27% by pretreatment with 2 muM GF 109203X to inactivate protein kinase C. We found that fMLP increase reactive oxygen intermediate (ROI) production in neutrophils. which can be suppressed by U73122 pretreatment. Collectively, this study shows that in human neutrophils, fMLP increased [Ca2+](i) concentration-dependently by re Leasing Ca2+ from phospholipase C-coupled. thapsigargin-sensitive stores. accompanied by Ca2+ entry. The fMLP-induced [Ca2+](i) rise was modulated by protein kinase C, and the fMLP-induced Ca2+ entry was abolished by La3+, and was reduced by econazole, SKF96365 and inhibition of phospholipase A(2). (C) 2001 Elsevier Science B.V. All rights reserved.